High Mobility Group Box 1 Pathway Research Articles

The cardioprotection induced by lipopolysaccharide involves phos-phoinositide 3-kinase/Akt and high mobility group box 1 pathways

Objective The mechanisms by which lipopolysaccharide (LPS) pretreatment induces cardioprotection following ischaemia/reperfusion (I/R) have not been fully elucidated. We hypothesized that activation of phosphoinositide 3-kinase (PI3K)/Akt and high mobility group box 1 (HMGBx1) signaling plays an important role in LPS-induced cardioprotection. Methods In in vivo experiments, age- and weight-matched male C57BL/10Sc wild type mice were pretreated with LPS before ligation of the left anterior descending coronary followed by reperfusion. Infarction size was examined by triphenyltetrazolium chloride (TTC) staining. Akt, phospho-Akt, and HMGBx1 were assessed by immunoblotting with appropriate primary antibodies. In situ cardiac myocyte apoptosis was examined by the TdT-mediated dUTP nick-end labeling (TUNEL) assay. In an in vitro study, rat cardiac myoblasts (H9c2) were subdivided into two groups, and only one was pretreated with LPS. After pretreatment, the cells were transferred into a hypoxic chamber under 0.5% O 2. Levels of HMGBx1 were assessed by immunoblot. Results In the in vivo experiment, pretreatment with LPS reduced the at risk infarct size by 70.6% and the left ventricle infarct size by 64.93% respectively. Pretreatment with LPS also reduced cardiac myocytes apoptosis by 39.1% after ischemia and reperfusion. The mechanisms of LPS induced cardioprotection involved increasing PI3K/Akt activity and decreasing expression of HMGBx1. In the in vitro study, pretreatment with LPS reduced the level of HMGBx1 in H9c2 cell cytoplasm following hypoxia. Conclusion The results suggest that the cardioprotection following I/R induced by LPS pretreatment involves PI3K/Akt and HMGBx1 pathways.

017 INHIBITORY INTERACTIONS BETWEEN HMGB1 AND HEME OXYGENASE-1 PATHWAYS IN OSTEOARTHRITIC SYNOVIOCYTES

Purpose: Extracellular high mobility group box 1 (HMGB1) has been demonstrated to participate in inflammatory processes in joint diseases. Activation of osteoarthritic synoviocytes by proinflammatory cytokines results in HMGB1 translocation and release. We have shown recently a potential modulation of HMGB1 by heme oxygenase-1 (HO-1). In this study, we have examined the interactions between both pathways in osteoarthritic synoviocytes. Methods: Synovial tissue samples were obtained from 15 osteoarthritic patients undergoing total knee joint replacement. Synoviocytes were obtained by digestion with collagenase and cultured until third passage. Cells were treated with human recombinant HMGB1 (15 ng/ml) in the presence or absence of IL-1β (100 U/ml). HO-1 was induced by cobalt protoporphyrin IX (CoPP). Lentiviral HO-1.flag vector was also used for HO-1 overexpression. HO-1 gene silencing was achieved by using a specific siRNA. Gene expression was analyzed by quantitative PCR and protein expression by Western Blot and ELISA. HMGB1 translocation into the cytoplasm was studied by immunofluorescence. Results: Treatment of synoviocytes with HMGB1 down-regulated HO-1. This effect was potentiated by IL-1β and accompanied by a significant increase in matrix metalloproteinase (MMP)-1 and MMP-3 gene expression and activity. Conversely, induction of HO-1 by CoPP in the presence of IL-1 β led to reduced expression of both protein and mRNA of HMGB1. These effects were accompanied by a significant reduction in gene expression of MMP-1 and MMP-3, and MMP activity. The consequences of HO-1 induction were counteracted by HO-1 gene silencing. Interestingly, we observed a marked reduction in the translocation of HMGB1 from the nucleus into the cytoplasm. Transfection with lentiviral HO-1.flag vector confirmed the inhibitory effect of HO-1 on HMGB1 translocation. Conclusions:We have provided direct evidence that HO-1 inhibits the translocation of HMGB1 into the cytoplasm. Our data indicate a reciprocal negative regulation between HO-1 and HMGB1 pathways in osteoarthritic synoviocytes.

Growth suppression and radiosensitivity increase by HMGB1 in breast cancer

HMGB1 (high-mobility group box-1) is a nuclear protein containing a consensus RB (retinoblastoma)-binding LXCXE motif. In this study, we studied the potential association of HMGB1 and RB and the in vitro and in vivo activities of HMGB1 in human breast cancer cells. The protein-protein interaction was determined by immunoprecipitation-Western blotting and glutathione-S-transferase capture assays; cell growth and radiosensitivity were examined by cell counts, MTT assay, and clonogenic assay; cell cycle progression and apoptosis were evaluated using flow cytometry; and the antitumor activity of HMGB1 was examined with tumor xenografts in nude mice. HMGB1 was associated with RB via a LXCXE motif-dependent mechanism. HMGB1 enhanced the ability of RB for E2F and cyclin A transcription repression. The increased expression of HMGB1 conferred an altered phenotypes characterized by the suppression of cell growth; G1 arrest and apoptosis was induced in MCF-7 cells containing the wild-type retinoblastoma (Rb) gene, but showed no activities in BT-549 cells containing the Rb gene deletion. The HMGB1-induced apoptosis accompanied by caspase 3 activation and PARP (poly(ADP-ribose)polymerase) cleavage. HMGB1 elevated the radiosensitivity of breast cancer cells in both the MCF-7 and BT-549 cell lines. The enhanced expression of HMGB1 caused a suppression of growth of MCF-7 tumor xenografts in nude mice, while LXCXE-defective HMGB1 completely lost antitumor growth activity. HMGB1 functions as a tumor suppressor and radiosensitizer in breast cancer. A HMGB1-RB interaction is critical for the HMGB1-mediated transcriptional repression, cell growth inhibition, G1 cell cycle arrest, apoptosis induction, and tumor growth suppression, but is not required for radiosensitization. Therefore, it may be possible to design new therapies for the treatment of breast cancer that exert their effects by modulating the HMGB1 and RB regulatory pathway and HMGB1-related gene therapy.